This application claims the benefit of Korean Patent Application No. 10-2005-0005537, filed on Jan. 20, 2005, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a cell lysis method using temperature variation, and more particularly, to a method of lysing cells using temperature variation through repeated cooling and heating processes, in which an endothermic reaction is used to cause temperature variations, which is convenient to perform and reduces time and costs.
2. Description of the Related Art
Cell lysis is a process of releasing materials in a cell by disrupting the cell membrane, and in particular, a process of extracting intracellular materials from a cell to isolate DNA or RNA before amplification, such as a polymerase chain reaction (PCR).
Cell lysis methods through cell rupture can be classified into mechanical methods and non-mechanical methods.
Mechanical methods include ultrasonication, disruption using a homogenizer, pressing using, for example, a French press, etc., decompression, pulverization, etc. Non-mechanical methods include chemical methods, thermal methods, enzymatic methods, etc.
For example, conventional ultrasonication, which is a mechanical method, is performed on cell solution and suspension in a chamber located in an ultrasonic bath. Such ultrasonic disruption leads to many disadvantages in cell lysis. First, a nonuniform ultrasonic energy distribution causes inconsistent results. Second, the ultrasonic bath cannot concentrate on the chamber, and thus, a large amount of time is required to complete cell disruption.
Chemical methods, which are non-mechanical methods, use, for example, an acid, a base, a detergent, a solvent, a chaotropic reagent, etc. Especially, a chemical method using a detergent is widely used. Detergents disrupt a lipid double membrane to release cell contents and lyses membrane protein. Detergents are most commonly used to lyse animal cells. Most detergents denature protein. However, a reagent for cell lysis should be separately added, and hence a subsequent process of removing the reagent is required. PCR inhibition may occurs, and the process takes a long time.
Enzymatic methods use lysozyme, protease, etc.
Thermal methods include freezing-thawing, heating, osmotic impact, electric impact, etc. For example, cell lysis is achieved by contacting cells with a hot object, such as a hot plate, or by repeating a cycle of freezing to −70° C. and thawing to room temperature.
A cell lysis method using heat, which is neither a mechanical method nor uses a reagent, is suitable to be used in a lab-on-a-chip (LOC). In particular, the freezing-thawing method is known to be more efficient to lyse cells than a simple heating method. However, an expensive rapid cooling apparatus for cooling to −70° C. is required and it takes a long time. Further, when a small amount of a sample is used as in a LOC, the moisture in the sample easily vaporizes and the sample easily dries. In addition, this method causes protein denaturation, which inhibits PCR. These problems also arise with simple heating methods.
In particular, when cell lysing gram positive bacteria, it is difficult to obtain a desired cell lysis effect only using a conventional heating method.
Thus, the inventors performed intensive research in order to discover a cell lysis method of repeating cooling and heating processes that does not require a separate expensive apparatus, can be used regardless of time and place, is simple and convenient to perform, and does not inhibit PCR, and completed the present invention.